Antihelminthic anthraquinones and method of use thereof

ABSTRACT

Anthraquinones are described which are antihelminthic and in particular, are useful in compositions for inhibiting  Schistosoma  sp. in vitro or in vivo. The preferred anthraquinones have the formula: 
                 
 
wherein R 1 , R 2 , R 3 , and R 4  are each hydrogen, hydroxy, halogen, alkyl, substituted alkyl, alkene, substituted alkene, alkyne, aryl, substituted aryl, cyclic, substituted cyclic, acid group, carbohydrate, or combination thereof, R is a group containing 1 to 12 carbons such as methyl, alkyl, substituted alkyl, aldehyde, hydroxy, hydroxymethyl, acid group, carbohydrate, or combination thereof, and the halogen is I, F, Br, or Cl.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of application Ser. No. 10/317,906 filedDec. 12, 2002 now U.S. Pat. No. 6,800,615 claims priority to ProvisionalApplication Ser. No. 60/372,576, filed Apr. 15, 2002, and ProvisionalApplication Ser. No. 60/389,368, filed Jun. 17, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

Reference to a “Computer Listing Appendix submitted on a Compact Disc”

Not Applicable.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to anthraquinones which are antihelminthicand in particular, are useful in compositions for inhibiting Schistosomasp. in vitro or in vivo. The preferred anthraquinones have the formula:

wherein R₁, R₂, R₃, and R₄ are each hydrogen, hydroxy, halogen, alkyl,substituted alkyl, alkene, substituted alkene, alkyne, aryl, substitutedaryl, cyclic, substituted cyclic, acid group, carbohydrate, orcombination thereof, R is a group containing 1 to 12 carbons such asmethyl, alkyl, substituted alkyl, aldehyde, hydroxy, hydroxymethyl, acidgroup, carbohydrate, or combination thereof, and the halogen is I, F,Br, or Cl. In a particular embodiment, the anthraquinones have theformula

wherein R is a group containing 1 to 12 carbons such as methyl, alkyl,substituted alkyl, aldehyde, hydroxy, hydroxymethyl, acid group,carbohydrate, and combinations thereof.

(2) Description of Related Art

Schistosomasis is a disease caused by parasitic digenetic trematodes ofthe genus Schistosoma that afflicts at least 200 million peopleworldwide with another 600 million at risk (Chitsula et al., Acta Trop.77: 41-51 (2000)). Chronic Schistosoma infection can lead to thedevelopment of a variety of conditions including diarrhea, hepaticfibrosis and portal hypertension, central nervous system disease,embolisms of the pulmonary arterioles, and hematuria. While a largenumber of schistosomes are known, only five appear to be primarilyresponsible for human infections including Schistosoma mansoni,Schistosoma japonicum, Schistosoma mekongi, Schistosoma intercalatum,and Schistosoma haematobium.

These digenetic schistosomes have a complex life-cycle in whichfree-swimming cercariae emerge from intermediate freshwater snail hostsand infect humans by attaching to the skin via an oral sucker or mucussecretion and penetrate the dermis by releasing proteolytic enzymes.Concurrently, the cercariae shed their tails and transform intoschistosomula that enter the venous vascular system where they arecarried to the heart and lungs before reaching the systemic circulation.Ultimately, the schistosomula arrive at the liver where they grow intosexually mature adults. Male and female adults form copulating pairsthat migrate down the portal vein, eventually reaching the mesenteric orvesical veins, depending on the specific species of schistosome, andbegin laying eggs for a period of typically 3 to 5 years. The eggs aregenerally responsible for triggering the host's immune response thatresults in the formation of granulomas that lead to the sequelae ofclinical manifestations (Bica et al., Infect. Dis. Clin. N. Am. 14:637-642 (2000); Elliot, Gastroenterol. Clin. N. Am. 25: 599-624 (1996);Morris and Knauer, Sem. Respir. Infect. 12: 159-170 (1997); Schafer andHale, Curr. Gastroenterol. Reports 3: 293-303 (2001)).

There are limited options available for the chemotherapeutic treatmentfor Schistosoma infections with the drug-of-choice being thepyrazionoisoquinoline, praziquantel (Elliot, ibid.). Unfortunately, thelong-term worldwide application of the drug coupled with the recentdiscovery of praziquantel-tolerant schistosomes has generated concernover the development of drug-resistant Schistosoma strains (Cioli,Parasitol. Today 14: 418-422 (1998) and Curr. Opin. Infect Dis. 13:659-663 (2000); William et al., Parasitol. 122: 63-66 (2001)). With fewother options available for combating schistosomiasis, there is anurgent need to develop new methodologies for the treatment andprevention of Schistosoma infection (Cioli, ibid.).

Daylily roots (Hemerocallis spp., Hemerocallidaceae) have been used inAsia to treat schistosomiasis (Shiao et al., Acta Pharma. Sinica9:218-224 (1962); Shiao et al., Acta Pharma. Sinica 9: 217-224 (1962)).However, this method of treatment has been disfavored due to a host oftoxic side effects and deaths associated with the administration ofHemerocallis root extracts to humans (Wang et al., Phytochem. 28:1825-1826 (1989)). Previous efforts to identify the active constituentresponsible for the therapeutic properties of Hemerocallis roots lead toisolation of a neurotoxic binaphthalenetetrol known as stypandrol (Wangand Yang, 1993) which had been shown to cause paralysis, blindness anddeath in mammals (Main et al., Aust. Vet. J. 57: 132-135 (1981);Colegate et al., Aust. J. Chem. 38: 1233-1241 (1985)). In another reportby Chen et al. (Acta Pharma. Sinica 9: 579-586 (1962)), researchersobtained a yellow powdery isolate to which the authors ascribed both thebiological activity against schistosomes, as well as, the toxic sideeffects associated with the use of Hemerocallis roots; however, itsstructure was never identified. While other studies have describedadditional compounds found in daylilies, none of these efforts haveaddressed the need to fully characterize the bioactive schistosomicidalchemical constituents from Hemerocallis roots.

Compounds which have antihelminthic activity are known in the prior artsuch as oxamniquine, metrifonate, and4-(4-nitroanilino)-phenylisothiocyanate, which are disclosed in U.S.Pat. No. 4,117,156 to Loewe et al. However, oxamniquine is onlyeffective against Schistosoma mansoni, is more active against malerather than female worms, and has little effect on immature worms, andmetrifonate is only active against Schistosoma haematobium. U.S. Pat.Nos. 5,091,385, 5,177,073, and 5,489,590 to Gulliya et al. disclose atherapeutic mixture comprising a photoactive compound which is capableof killing tumors, bacteria, viruses, and parasites such as Schistosomawhen activated prior to use with an activating agent such as a chemical,radiation (preferably, irradiation with a laser), gamma rays, orelectrons from an electropotential device. The photoactive compoundsinclude a general suggestion of anthraquinones.

In light of the above, there remains a need for novel compounds withantihelminthic activity to increase the arsenal of drugs for combatinghelminthic infections in warm-blooded animals, including humans.

SUMMARY OF THE INVENTION

The present invention provides a method for inhibiting helminths such asthose comprising the Schistosoma genus in vivo or in vitro by exposingthe helminths to an inhibitory amount of one or more anthraquinones. Theanthraquinones can be substituted with halogens such as I, F, Br, and Clin the ring, particularly where hydroxyl groups are not located. Thesubstituents in the ring can also include one or more of the halogens.

As used herein, the term “inhibitory” means either to limit the growthof the helminth or cells, to stop the growth of the helminth or cells,or to kill the helminth or cells. Thus, the term embraces any affectwhich adversely affects the helminth or cells.

Therefore, in one embodiment, the present invention provides a methodfor inhibiting a parasitic helminth which comprises exposing thehelminth to an inhibitory amount of an anthraquinone.

In particular, the present invention provides a method for inhibiting aparasitic helminth, which comprises exposing the helminth to anantihelminthic amount of at least one anthraquinone which has theformula:

wherein R₁, R₂, R₃, and R₄ are each selected from the group consistingof hydrogen, hydroxy, halogen, alkyl, substituted alkyl, alkene,substituted alkene, alkyne, aryl, substituted aryl, cyclic, substitutedcyclic, acid group, carbohydrate, and combinations thereof, R is a groupcontaining 1 to 12 carbons selected from the group consisting of methyl,alkyl, substituted alkyl, aldehyde, hydroxy, hydroxymethyl, acid group,carbohydrate, and combinations thereof, and the halogen is selected fromthe group consisting of I, F, Br, and Cl.

The present invention further provides a method for inhibiting aparasitic helminth, which comprises exposing the helminth to anantihelminthic amount of at least one anthraquinone which has theformula:

wherein R is a group containing 1 to 12 carbons selected from the groupconsisting of methyl, alkyl, substituted alkyl, aldehyde, hydroxy,hydroxymethyl, acid group, carbohydrate, and combinations thereof.

The present invention further provides a method for inhibiting aSchistosoma sp. which comprises exposing the Schistosoma sp. to aninhibitory amount of at least one anthraquinone which has the formula:

wherein R₁, R₂, R₃, and R₄ are each selected from the group consistingof hydrogen, hydroxy, halogen, alkyl, substituted alkyl, alkene,substituted alkene, alkyne, aryl, substituted aryl, cyclic, substitutedcyclic, acid group, carbohydrate, and combinations thereof, R is a groupcontaining 1 to 12 carbons selected from the group consisting of methyl,alkyl, substituted alkyl, aldehyde, hydroxy, hydroxymethyl, acid group,carbohydrate, and combinations thereof, and the halogen is selected fromthe group consisting of I, F, Br, and Cl.

The present invention further provides a method for inhibiting aSchistosoma sp. which comprises exposing the Schistosoma sp. to aninhibitory amount of at least one anthraquinone of the formula:

wherein R is a group containing 1 to 12 carbons selected from the groupconsisting of methyl, alkyl, substituted alkyl, aldehyde, hydroxy,hydroxymethyl, acid group, carbohydrate, and combinations thereof.

In a preferred embodiment of the above methods, the anthraquinone is1,2,8-trihydroxy-3-methyl anthraquinone (compound 3),1,2,8-trihydroxy-3-hydroxymethyl anthraquinone (compound 6), or both andthe inhibiting can be either in vivo or in vitro.

In a further embodiment of the above methods, the anthraquinone isinhibitory at a dosage of 1 to 1,000 micrograms per milliliter or gram.

The present invention further provides a method for inhibiting apathogenic trematode in a warm-blooded animal or human infected with thepathogenic trematode comprising (a) providing a composition containingan inhibitory amount of at least one anthraquinone selected from thegroup consisting of 1,2,8-trihydroxy-3-methyl anthraquinone (compound 3)and 1,2,8-trihydroxy-3-hydroxymethyl anthraquinone (compound 6) in apharmaceutically acceptable carrier; and (b) and administering thecomposition to the warm-blooded animal or human to inhibit thepathogenic trematode. A particular composition is a topical compositionfor swimmers itch which is a species of Schistosoma.

In a further embodiment of the method, the anthraquinone is inhibitoryat a dosage of 1 to 1,000 micrograms per milliliter or gram.

In a further still embodiment of the method, the anthraquinone isadministered to the warm-blooded animal or human orally, subcutaneously,intraperitoneally, topically, intravenously, topically, intranasally, orrectally.

The present invention further provides a method for inhibiting apathogenic trematode in a warm-blooded animal or human infected with thepathogenic trematode comprising (a) providing a composition containingan inhibitory amount of 1,2,8-trihydroxy-3-methyl-O-β-D-glucopyranosideanthraquinone (compound 7) and at least one anthraquinone selected fromthe group consisting of 1,8-dihydroxy-2-O-β-D-glucopyranosideanthraquinone (compound 4) and1,8-dihydroxy-2-O-malonyl-(1-6)-β-D-glucopyranoside anthraquinone(compound 5) in a pharmaceutically acceptable carrier; and (b) andadministering the composition to the warm-blooded animal or human toinhibit the pathogenic trematode.

In a further embodiment of the method, the composition further includesan inhibitory amount of at least one anthraquinone selected from thegroup consisting of 1,2,8-trihydroxy-3-methyl anthraquinone (compound 3)and 1,2,8-trihydroxy-3-hydroxymethyl anthraquinone (compound 6).

In a further still embodiment of the method, the anthraquinone isinhibitory at a dosage of 1 to 1,000 micrograms per milliliter or gram.

In a still further embodiment, the anthraquinone is administered to thewarm-blooded animal or human orally, subcutaneously, intraperitoneally,topically, intranasally, intravenously, or rectally.

In a preferred embodiment of the above methods, the anthraquinone isselected from the group consisting of 1-hydroxy-2-acetyl-3,6-methylanthraquinone (compound 1), 2-acetyl-3,6-methyl anthraquinonemonoacetate (compound 1a), 1-hydroxy-2-acetyl-3,7-methyl anthraquinone(compound 2), 2-acetyl-3,7-methyl anthraquinone monoacetate (compound2a), 1,2,8-trihydroxy-3-methyl anthraquinone (compound 3),1,8-dihydroxy-2-O-β-D-glucopyranoside anthraquinone (compound 4),1,2,8-trihydroxy-3-hydroxymethyl anthraquinone (compound 6), and1,8-dihydroxy-3-carboxy anthraquinone (compound 8) and the inhibitingcan be either in vivo or in vitro.

In a further embodiment of the above methods, the anthraquinone isinhibitory at a dosage of 1 to 1,000 micrograms per milliliter or gram.

The present invention further provides an antihelminthic compositionwhich comprises (a) at least one anthraquinone which has the formula:

wherein R₁, R₂, R₃, and R₄ are each selected from the group consistingof hydrogen, hydroxy, halogen, alkyl, substituted alkyl, alkene,substituted alkene, alkyne, aryl, substituted aryl, cyclic, substitutedcyclic, acid group, carbohydrate, and combinations thereof, R is a groupcontaining 1 to 12 carbons selected from the group consisting of methyl,alkyl, substituted alkyl, aldehyde, hydroxy, hydroxymethyl, acid group,carbohydrate, and combinations thereof, and the halogen is selected fromthe group consisting of I, F, Br, and Cl; and (b) a pharmaceuticallyacceptable carrier, preferably wherein the composition contains betweenabout 1 and 1,000 micrograms of the anthraquinone per milliliter or gramof the carrier.

Preferably, the anthraquinone has the formula:

wherein R is a group containing 1 to 12 carbons selected from the groupconsisting of methyl, alkyl, substituted alkyl, aldehyde, hydroxy,hydroxymethyl, acid group, carbohydrate, and combinations thereof.

More preferably, the anthraquinone is selected from the group consistingof 1-hydroxy-2-acetyl-3,6-methyl anthraquinone (compound 1),2-acetyl-3,6-methyl anthraquinone monoacetate (compound 1a),1-hydroxy-2-acetyl-3,7-methyl anthraquinone (compound 2),2-acetyl-3,7-methyl anthraquinone monoacetate (compound 2a),1,2,8-trihydroxy-3-methyl anthraquinone (compound 3),1,8-dihydroxy-2-O-β-D-glucopyranoside anthraquinone (compound 4),1,2,8-trihydroxy-3-hydroxymethyl anthraquinone (compound 6), and1,8-dihydroxy-3-carboxy anthraquinone (compound 8).

The present invention also provides an isolated and purifiedanthraquinone which has the formula:

The present invention also provides an isolated and purifiedanthraquinone which has the formula:

The present invention also provides an isolated and purifiedanthraquinone which has the formula:

The present invention also provides an isolated and purifiedanthraquinone which has the formula:

The present invention also provides an isolated and purifiedanthraquinone which has the formula:

The present invention also provides an isolated and purifiedanthraquinone which has the formula:

The present invention also provides an isolated and purifiedanthraquinone which has the formula:

The present invention also provides an isolated and purifiedanthraquinone which has the formula:

The present invention also provides an isolated and purifiedanthraquinone which has the formula:

OBJECTS

Therefore, it is an object of the present invention to providecompositions such as the anthraquinones disclosed herein which haveantihelminthic activity.

It is further an object of the present invention to provide methods forusing the anthraquinones to inhibit helminths infecting warm-bloodedanimals, including humans.

Further still, it is an object of the present invention to providemethods for using the anthraquinones to inhibit pathogenic trematodessuch as those of the Schistosoma genus infecting warm-blooded animals,including humans.

These and other objects of the present invention will becomeincreasingly apparent with reference to the following drawings andpreferred embodiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing the chemical structure of compounds 1 to 12isolated from daylily roots of Hemerocallis fulva “Kwanzo” Kaempfer(1712). Ac refers to acetyl groups such as —COCH₃.

FIG. 2A shows the difference NOE (→) and long-range COSY (-)correlations used to establish the structure of compound 1(kwanzoquinone A).

FIG. 2B shows the difference NOE (→) and long-range COSY (-)correlations used to establish the structure of compound 2(kwanzoquinone B).

FIG. 3 shows selected HMBC correlations used to determine the structureof compound 5 (kwanzoquinone D).

FIG. 4 shows selected HMBC correlations used to determine the structureof compound 6 (kwanzoquinone E).

FIG. 5 shows selected HMBC correlations used to determine the structureof compound 11 (5-hydroxydianellin).

FIG. 6 shows the dose response effect of compound 3(2-hydroxychrysophanol) and compound 6 (kwanzoquinone E) on S. mansonicercariae mobility. Mobility was accessed bases on the movement andswimming behavior of the invasive aquatic larval stage.

FIG. 7 is a graph showing the percent inhibition of mobilization ofcercariae as a function of concentration of compounds 3 (B) and 6 (F).

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

The present invention provides anthraquinones and methods for their useas antihelminthic compounds to combat helminthic infections ofwarm-blooded animals, including humans. In particular, theanthraquinones are hydroxy-anthraquinones, which along withanthraquinones in general, are believed to be unknown in the prior artas being useful per se for antihelminthic applications.Hydroxy-substituted anthraquinones can be derived synthetically asdescribed by Khan et al., in Synthesis 255-257 (1994) and by Cameron etal. in Tetrahedron Letters 27: 4999-5002 (1986) or can be isolated fromplant sources such as the roots of daylilies (Hemerocallis fulva) asdescribed hereinafter.

As described herein in the Examples, the roots of H. fulva (Kwanzo) wereextracted with hexane, EtOAc, and MeOH. The hexane and MeOH extractswere selected for further study and subsequently subjected to acombination of chromatographic procedures including Si gel MPLC andPTLC, ODS MPLC and preparative HPLC, and crystallization. This led tothe discovery and isolation of nine novel anthraquinones, thekwanzoquinones: kwanzoquinone A (compound 1:(1-hydroxy-2-acetyl-3,6-methyl anthraquinone), kwanzoquinone Amonoacetate (compound 1a: (2-acetyl-3,6-methyl anthraquinonemonoacetate), kwanzoquinone B (compound 2:(1-hydroxy-2-acetyl-3,7-methylanthraquinone), kwanzoquinone B monoacetate (compound 2a:(2-acetyl-3,7-methyl anthraquinone monoacetate), kwanzoquinone C(compound 4: (1,8-dihydroxy-2-O-β-D-glucopyranoside anthraquinone),kwanzoquinone D (compound 5:(1,8-dihydroxy-2-malonyl-(1→6)—O-β-D-glucopyranoside anthraquinone),kwanzoquinone E (compound 6: (1,2,8-trihydroxy-3-hydroxymethylanthraquinone), kwanzoquinone F (compound 7:(1,2,8-trihydroxy-3-methyl-O-β-D-glucopyranoside anthraquinone), andkwanzoquinone G (compound 9: (1,8-dihydroxy-2-methyl-3-carboxyanthraquinone) and a novel naphthalene glycoside (compound 11;5-hydroxydianellin). The structures and complete ¹H and ¹³C NMR spectralassignments for these novel compounds, as well as those for knowncompounds 3 (an anthraquinone known as1,2,8-hydroxy-3-methylanthraquinone or 2-hydroxychrysophanol), 10(dianellin), and 12 (6-methylluteolin), were made based on thorough 1Dand 2D NMR studies and are disclosed herein for the first time. Thestructures of the above compounds are shown in FIG. 1. Theanthraquinones are soluble in a variety of protic and aprotic solventsincluding, but not limited to, DMSO, alcohols such as ethanol, aqueousalkali hydroxide solutions, Na₂CO₃ solutions, and NH₃ solutions.

When the anthraquinones were tested for antihelminthic activity, it wasdiscovered that compounds 3 and 6 had antihelminthic activity. Compound3 at a concentration of about 25 μg/mL was found to rapidly immobilizeSchistosoma cercariae within about 15 seconds of exposure and to killabout 50% of the cercariae by 24 hours post-exposure. At a concentrationof about 3.125 μg/mL, the cercariae were immobilized within about 45minutes of exposure. Compound 6 at a concentration of about 25 μg/mL wasalso found to immobilize the cercariae but over a time frame of about 12to 14 minutes. However, compound 6 killed all of the cercariae by 24hours post-exposure. Compounds 4 and 5 are hydrolyzable to compound 3and compound 7 is hydrolyzable to compound 6. These results demonstratethat the anthraquinones, particularly compounds 3 and 6, have differentmodes of action but that both are useful to treat helminthic infectionseither separately or in combination.

Therefore, the anthraquinones of the present invention, which are usefulas antihelminthic compounds, include both the particular anthraquinonescompounds with the antihelminthic activity per se and anthraquinonescompounds which are hydrolyzed in the gut of the helminth orwarm-blooded animal, including humans, or hydrolyzed in vitro to producethe compounds with the antihelminthic activity. For example,anthraquinones with sugar substituents (compounds 4, 5, and 7) can behydrolyzed in the gut of the helminth or warm-blooded animals, includinghumans, to which the compounds are administered to produce theanthraquinones with antihelminthic activity.

The anthraquinones are particularly useful in a method of treatment forinhibiting helminths, particularly those helminths which are importantin human medicine. Such helminths include those which reside in theintestinal tract such as hookworms, particularly Ancyclostoma orNecator, and those which reside in the bloodstream such as the parasiticdigenetic trematodes of the genus Schistosoma, particularly Schistosomahaematobium, Schistosoma mansoni, Schistosoma mekongi, Schistosomaintercalatum, and Schistosoma japonicum.

The anthraquinones which have antihelminthic activity and, therefore,are useful as antihelminthic compounds have the following generalchemical formula:

wherein R₁, R₂, R₃, and R₄ are each selected from the group consistingof hydrogen, hydroxy, halogen, alkyl, substituted alkyl, alkene,substituted alkene, alkyne, aryl, substituted aryl, cyclic, substitutedcyclic, acid group, carbohydrate, and combinations thereof, R is a groupcontaining 1 to 12 carbons selected from the group consisting of methyl,alkyl, substituted alkyl, aldehyde, hydroxy, hydroxymethyl, acid group,carbohydrate, and combinations thereof, and the halogen is selected fromthe group consisting of I, F, Br, and Cl.

In a particular embodiment, the anthraquinones have the followinggeneral chemical structure:

wherein R is a group containing 1 to 12 carbons selected from the groupconsisting of methyl, alkyl, substituted alkyl, aldehyde, hydroxy,hydroxymethyl, acid group, carbohydrate, and combinations thereof.

The preferred anthraquinones with antihelminthic activity with the abovegeneral chemical formula are 1,2,8-trihydroxy-3-methylanthraquinone,which is compound 3 isolated from daylilies and which has the trivialname 2-hydroxychrysophanol, and which has the chemical formula:

and 1,2,8-trihydroxy-3-hydroxymethylanthraquinone, which is novelcompound 6 isolated from daylilies and which has been given thetrivial-name kwanzoquinone F, and which has the chemical formula:

Anthraquinones which can be hydrolyzed in vitro or in vivo such as inthe gut of helminths or warm-blooded animals or humans to anthraquinoneswith antihelminthic activity are also useful as antihelminthiccompounds. These anthraquinones have the general chemical formula A:

or the general chemical formula B:

wherein for each, R is selected from the group consisting of loweralkyl, lower substituted alkyl containing 1 to 12 carbon atoms, analdehyde group, a carbohydrate, and an acid group.

The preferred anthraquinones which can be hydrolyzed to anthraquinoneswith antihelminthic activity with the general chemical formula A include1,8-dihydroxy-2-O-β-glucopyranoside-3-methylanthraquinone, which isnovel compound 4 isolated from daylilies and which has been given thetrivial name kwanzoquinone D, and which has the chemical formula:

and 1,8-dihydroxy-2-Oβ-D-glucopyranoside ((1-6)malonyl)-3-methylanthraquinone, which is novel compound 5 isolated fromdaylilies and which has been given the trivial name Kwanzoquinone E, andwhich has the chemical formula:

The preferred anthraquinone which can be hydrolyzed to a anthraquinonewith antihelminthic activity with the general chemical formula B is1,2,8trihydroxy-3-methyl-O-β-D-glucopyranoside-anthraquinone, which isnovel compound 7 isolated from daylilies and which has been given thetrivial name kwanzoquinone F, and which has the chemical formula:

As is evident from the chemical formulas for compound 4 and 5, wheneither compound is hydrolyzed in vivo or in vitro, it is hydrolyzed tocompound 3. When compound 7 is hydrolyzed in vivo or in vitro, it ishydrolyzed to compound 6. As shown herein, both compounds 3 and 6 haveantihelminthic activity.

The method for treating a warm-blooded animal or human infected with ahelminth (patient), in particular a pathogenic trematode such asSchistosoma sp., comprises providing to the warm-blooded animal or humanan antihelminthic composition comprising as the active ingredient aninhibitory amount of one or more of the anthraquinones, preferably oneor more anthraquinones selected from the group consisting of compounds3, 4, 5, 6, and 7.

For example, in one embodiment, the warm-blooded animal or human isprovided an inhibitory amount of compound 3 or compound 6. In a furtherembodiment, which is preferred, the warm-blooded animal or human isprovided an inhibitory amount of compounds 3 and 6. In an embodimentfurther still, the warm-blooded animal or human is provided aninhibitory amount of at least one compound selected from the groupconsisting of compounds 4, 5, and 7. In an embodiment further still, thewarm-blooded animal or human is provided an inhibitory amount ofcompound 7 and inhibitory amount of at least one compound selected fromthe group consisting of compounds 4 and 5. In an embodiment furtherstill, the warm-blooded animal or human is provided an inhibitory amountof compound 3 and an inhibitory amount of compound 7. In an embodimentfurther still, the warm-blooded animal or human is provided aninhibitory amount of compound 7 and an inhibitory amount of at least onecompound selected from the group consisting of compounds 4 and 5 and aninhibitory amount of at least one compound selected from the groupconsisting of compounds 3 and 6. It is readily apparent that otherembodiments comprising particular combinations of the aforementionedcompounds can be used as the active ingredient in antihelminthiccompositions for inhibiting helminths, in particular pathogenictrematodes such as those of the Schistosoma genus. In the aforementionedcompositions, the anthraquinone is inhibitory at a dosage of 1 to 1,000micrograms per milliliter or gram.

Because the anthraquinones isolated from the day lily are antihelminthicwithout the need for activation prior to administering to thewarm-blooded animal or human, antihelminthic compositions comprising theanthraquinones can include a wide variety of embodiments foradministering the anthraquinones to the warm-blooded animal or human.Furthermore, the antihelminthic compositions can be administered towarm-blooded animals or humans in non-medical environments (outsidehospitals and medical clinics) and in environments where access toactivating agents is either limited, expensive, or non-existent.

In a preferred embodiment, one or more of the anthraquinones for curinga warm-blooded animal, including humans, of a helminth infection, orinhibiting the infection, are provided to the warm-blooded animal orhuman at an inhibitory dose in a pharmaceutically acceptable carrier.Therefore, the anthraquinones are processed with pharmaceutical carriersubstances by methods well known in the art such as by means ofconventional mixing, granulating, coating, suspending and encapsulatingmethods, into the customary preparations for oral, rectal, or other modeof administration. For example, antihelminthic anthraquinonepreparations for oral application can be obtained by combining one ormore of the anthraquinones with solid pharmaceutical carriers;optionally granulating the resulting mixture; and processing the mixtureor granulate, if desired and/or optionally after the addition ofsuitable auxiliaries, into the form of tablets or dragee cores.

Suitable pharmaceutical carriers for solid preparations are, inparticular, fillers such as sugar, for example, lactose, saccharose,mannitol or sorbitol, cellulose preparations and/or calcium phosphates,for example, tricalcium phosphate or calcium hydrogen phosphate; alsobinding agents, such as starch paste, with the use, for example, ofmaize, wheat, rice or potato starch, gelatine, tragacanth, methylcellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl celluloseand/or polyvinylpyrrolidone, esters of polyacrylates orpolymethacrylates with partially free functional groups; and/or, ifrequired, effervescent agents, such as the above-mentioned starches,also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, oralginic acid or a salt thereof, such as sodium alginate. Auxiliaries areprimarily flow-regulating agents and lubricating agents, for example,silicic acid, talcum, stearic acid or salts thereof, such as magnesiumstearate or calcium stearate. Dragee cores are provided with suitablecoatings, optionally resistant to gastric juices, whereby there areused, inter alia, concentrated sugar solutions optionally containing gumarabic, talcum, polyvinylpyrrolidone, and/or titanium dioxide, lacquersolutions in aqueous solvents or, for producing coatings resistant tostomach juices, solutions of esters of polyacrylates orpolymethacrylates having partially free functional groups, or ofsuitable cellulose preparations such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate, with or without suitablesofteners such as phthalic acid ester or triacetin. Dyestuffs orpigments may be added to the tablets or dragee coatings, for example foridentification or marking of the various doses of active ingredient.

Further antihelminthic preparations comprising one or more of theanthraquinones which can be administered orally are hard gelatinecapsules, as well as hard or soft closed capsules made from gelatineand, if required, a softener such as glycerin or sorbitol. The hardgelatine capsules can contain one or more of the anthraquinones in theform of a granulate, for example in admixture with fillers such as maizestarch, optionally granulated wheat starch, binders or lubricants suchas talcum, magnesium stearate or colloidal silicic acid, and optionallystabilizers. In closed capsules, the one or more of the anthraquinonesis in the form of a powder or granulate; or it is preferably present inthe form of a suspension in suitable solvent, whereby for stabilizingthe suspensions there can be added, for example, glycerin monostearate.

Other antihelminthic preparations to be administered orally are, forexample, aqueous suspensions prepared in the usual manner, whichsuspensions contain the one or more of the anthraquinones in thesuspended form and at a concentration rendering a single dosesufficient. The aqueous suspensions either contain at most small amountsof stabilizers and/or flavoring substances, for example, sweeteningagents such as saccharin-sodium, or as syrups contain a certain amountof sugar and/or sorbitol or similar substances. Also suitable are, forexample, concentrates or concentrated suspensions for the preparation ofshakes. Such concentrates can also be packed in single-dose amounts.

Suitable antihelminthic preparations for rectal administration are, forexample, suppositories consisting of a mixture of one or more of theanthraquinones with a suppository foundation substance. Such substancesare, in particular, natural or synthetic triglyceride mixtures. Alsosuitable are gelatine rectal capsules consisting of a suspension of theone or more of the anthraquinones in a foundation substance. Suitablefoundation substances are, for example, liquid triglycerides, of higheror, in particular, medium saturated fatty acids.

Likewise of particular interest are preparations containing the finelyground one or more of the anthraquinones, preferably that having amedian of particle size of 5 μm or less, in admixture with a starch,especially with maize starch or wheat starch, also, for example, withpotato starch or rice starch. They are produced preferably by means of abrief mixing in a high-speed mixer having a propeller-like, sharp-edgedstirring device, for example with a mixing time of between 3 and 10minutes, and in the case of larger amounts of constituents with coolingif necessary. In this mixing process, the particles of the one or moreof the anthraquinones are uniformly deposited, with a continuingreduction of the size of some particles, onto the starch particles. Themixtures mentioned can be processed with the customary, for example, theaforementioned, auxiliaries into the form of solid dosage units; i.e.,pressed for example into the form of tablets or dragees or filled intocapsules. They can however also be used directly, or after the additionof auxiliaries, for example, pharmaceutically acceptable wetting agentsand distributing agents, such as esters of polyoxyethylene sorbitanswith higher fatty acids or sodium lauryl sulphate, and/or flavoringsubstances, as concentrates for the preparation of aqueous suspensions,for example, with about 5- to 20-fold amount of water. Instead ofcombining the anthraquinone/starch mixture with a surface-activesubstance or with other auxiliaries, these substances may also be addedto the water used to prepare the suspension. The concentrates forproducing suspensions, consisting of the one or more of theanthraquinones/starch mixtures and optionally auxiliaries, can be packedin single-dose amounts, if required in an airtight and moisture-proofmanner.

In addition, the antihelminthic preparations can be administeredintraperitoneally, intranasally, subcutaneously, or intravenously. Ingeneral, for intraperitoneal, intranasal, subcutaneous, or intravenousadministration, one or more of the antihelminthic antroquinones areprovided by dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives. Preferably, the one or moreantihelminthic anthraquinones are provided in a composition acceptablefor intraperitoneal, subcutaneous, or intravenous use in warm-bloodedanimals, and humans in particular.

Antihelminthic preparations according to the present invention compriseone or more of the anthraquinones at a concentration suitable foradministration to warm-blooded animals, and humans in particular, whichconcentration is, depending on the mode of administration, between about0.3% and 95%, preferably between about 2.5% and 90%. In the case ofsuspensions, the concentration is usually not higher than 30%,preferably about 2.5%; and conversely in the case of tablets, drageesand capsules with the one or more of the anthraquinones, theconcentration is preferably not lower than about 0.3%, in order toensure an easy ingestion of the required doses of the one or moreanthraquinones. The treatment of warm-blooded animals or humans infestedwith parasitic helminths with the preparations comprising one or more ofthe anthraquinones is carried out preferably by a single oral, rectal,intraperitoneal, intranasal, subcutaneous, or intravenous administrationof an amount which contains a dose of the one or more anthraquinonessufficient to practically completely free the warm-blooded animal orhuman from the parasitic helminths, that is to say, an amount which issufficient of cure the warm-blooded animal or human of the infectioncaused by the parasitic helminths or inhibit the growth of the parasitichelminth in the warm-blooded animal or human. If required, this curativedose can be divided into several partial doses which are administered atintervals of several hours to several days. The administered dose of theone or more anthraquinones, is dependent both on the species and generalcondition of the warm-blooded animal or human to be treated and on thegenus and species of the helminths infecting the warm-blooded animal orhuman.

The anthraquinones are useful for curing warm-blooded animals and humansof infections, or inhibiting the infections, of Ascaridia galli,Trichostrongylidae, for example, Nippostrongylus brasiliensis orNematospiroides dubius, Ancylostomatidae, for example, Necatoramericanus and Ancylostoma ceylanicum, and Strongylidae; against Cestodasuch as Hymenolepsis nana, Anoplocephalidae and Taeniidae; andparticularly against Trematoda such as Fasciolida, for example, Fasciolahepatica, and particularly Schistosoma, for example, Schistosomamansoni, Schistosoma japonicum, Schistosoma mekongi, Schistosomaintercalatum, and Schistosoma hematobium; also against the pathogens offilariasis, for example, Dipetalonema witei and Litomosoides carinii.The antihelminthic preparations according to the invention can be used,therefore, for the treatment of warm-blooded animals and humans in thecase of infestation with parasitic helminthes such as theaforementioned, especially for the treatment of warm-blooded animals andhumans affected by schistosomiasis, hookworm infestation or filariasis.

The anthraquinones are also useful for treating fresh water toimmobilize and/or kill pathogenic helminths, particularly Schistosomacercariae, which are in the water. Thus, the anthraquinones are usefulin eradication programs for reducing the number of Schistosoma in oreliminating Schistosoma from fresh water lakes, ponds, streams, rivers,pools, and the like. In general, a solution comprising one or more ofthe anthraquinones is applied to body of water by spraying to thesurface or by injecting into the body of water below the surface.Alternatively, the one or more anthraquinones are applied to the body ofwater in a dry form.

The following examples are intended to promote a further understandingof the present invention.

EXAMPLE 1

This example illustrates the extraction and isolation of thekwanzoquinones from daylilies.

Hemoricallis fulva (Kwanzo) plants were purchased from the PerennialPatch (Wade, N.C.) in August 1999. The plants were grown on the MichiganState University Campus before being harvested in April 2001. The leaveswere removed and the roots and crowns of 124 plants were washed andfrozen at −4° C. The frozen roots were lyophilized and ground in aWARING blender yielding 2.2 kg of fine light-brown powder.

For isolation and purification of compounds 1 to 12 involved the use ofSEPHADEX LH-20 (Sigma-Aldrich, St. Louis, Mo.), Si gel (particle size40-63 μm) from Fischer Scientific (Pittsburgh, Pa.), AMBERLITE XAD-16resin from Supelco (Bellafonte, Pa.), LC-SORB SP-A-ODS gel (particlesize 25-40 pm) from Dychrom (Santa Clara, Calif.), and Si gel PTLCplates (20×20 cm; 250, 500, and 1000 pm thick) from Analtech, Inc.(Newark, Del.). Preparative HPLC was performed on a Japan AnalyticalIndustry Co. model LC-20 recycling preparative HPLC with a JAIGEL-C₁₈column (10 μm, 20 mm×250 mm). All solvents and chemicals were purchasedfrom Spectrum Laboratory Products, Inc. (New Brunswick, N.J.) and wereof ACS analytical grade.

The lyophilized roots (2.0 kg) were sequentially extracted with 3×8 Lportions of hexane ethyl acetate, and methanol yielding 25, 23, and 130g of extract, respectively. The hexane extract was redissolved in 500 mLof hexane and partitioned with 3×500 mL portions of methanol. Themethanol fractions were pooled yielding 15 g of extract which wasapplied to Si gel VLC and eluted with 4 L hexane, 3 L hexane-acetone(9:1), and 3 L hexane-acetone (3:2). The hexane elute (8.5 g) wassubjected to Si gel MPLC under gradient conditions with 100% hexane to100% acetone and 200 mL fractions were collected. All fractions wereanalyzed by TLC and pooled according to similarities in their profilesyielding fractions A1 to A4.

The hexane-acetone (9:1) eluate from the Si gel VLC was subjected to Sigel MPLC under gradient conditions with 100% hexane to hexane-acetone(1:1) providing fractions B1 to B4.

Fraction B2 (1.5 g) was rechromatographed by Si gel MPLC under gradientconditions-with 100% hexane to 100% EtOAc and 200 mL fractions werecollected and pooled based on TLC profiles giving fractions C1 to C4.

Fractions A3 (1 g), A4 (1 g), C2 (300 mg), and C3 (300 mg) were pooledbased further examination by TLC and applied to Si gel MPLC. Elution wascarried out under gradient conditions with 100% hexane to 100% CHCl₃ toCHCl₃-ethanol (1:1) 18 mL fractions D1 to D90 were collected.

Fractions D1 to D10 were pooled (500 mg) and further subjected to Si gelMPLC under gradient conditions with 100% hexane to hexane-acetone (97:3)and 15 mL fractions E1 to E40 were collected.

Fractions E6 to E20 (200 mg) were composed of primarily one majorcomponent and thus pooled and subjected to sequential Si gel PTLC withhexane-EtOAc (10:1) (72 mg), hexane-diethyl ether (6:1) (51 mg), andbenzene-CHCl₃ (20:1) yielding 30 mg of α-tocopherol as a clear oil thatexhibited spectral characteristics identical to those reported in theliterature (Baker and Myers, Pharmacol. Res. 8: 763-770 (1991)).

Fractions D12 to D45 (300 mg) were combined, applied to Si gel PTLCplates, and developed in benzene-CHCl₃ (10:1) twice. A bright yellowband (44 mg) was obtained and following extraction from the Si gel, itwas dissolved in a minimal volume of CHCl₃ and hexane added drop-wiseuntil a slight degree of turbidity was noted. The solution was stored at−20° C. yielding an inseparable 1:1 mixture (based on ¹H NMR) ofcompounds 1 and 2 as fine yellow needles (12 mg). Both compounds 1 and 2and their mono acetates 1a and 2a (prepared from Ac₂o/pyridine) weresubjected to a variety of chromatographic techniques including furtherSi gel TLC and MPLC, as well as, ODS MPLC and ODS preparative HPLC, butfailed to separate these two compounds.

The MeOH extract of the roots was dissolved in 800 mL MeOH—H₂O (3:1) andleft at 4° C. until a precipitate formed. The mixture was centrifuged(16,000×g, 15 min, 4° C.) and the supernatant decanted to give 30 g ofextract. This was applied to a column of XAD-16 resin and eluted with 10L H₂O, 6 L 25% aqueous MeOH, and 8 L 100% MeOH. The MeOH eluate (18 g)was dissolved in 500 mL H₂O and partitioned with CHCl₃ (3×300 mL). TheCHCl₃ fractions were pooled and dried yielding 2 g of extract that wasapplied to ODS MPLC and eluted with 50 to 100% MeOH and 16 mL fractionsF1 to F166 were collected. Fractions F116 to F125 were pooled giving 100mg of residue that was dissolved in MeOH-acetone (3:1) and stored at−20° C. yielding 7 mg of compound 8 as a yellow powder. Compound 8 wasidentified as rhein based on comparisons of its physical and spectraldata to those reported in the literature (Danielsen and Aksnes, Magn.Reson. Chem. 30: 359-360 (1992)).

The aqueous phase (16 g) from partitioning with CHCl₃ was dissolved in50 mL of MeOH and 450 mL of acetone was slowly added while stirring andthe mixture left at 4° C. The supernatant (14 g) was applied to ODS MPLCand eluted with 45 to 100% MeOH under gradient conditions yielding 750mL fractions G1 to G6. Fraction G3 (1 g) was again applied to ODS MPLCand eluted with CH₃CN-MeOH—H₂O-TFA (25:25:50:0.1 to 30:30:40:0.1) undergradient conditions yielding fractions H1 to H6. Fraction H5 (170 mg)was applied to SEPHADEX LH-20 with MeOH. The major component eluted as ayellow band (25 mg) and was further purified by ODS preparative HPLCwith CH₃CN—MeOH—H₂O-TFA (50:20:30:0.1) yielding 16 mg of compound 9 as ayellow powder.

Fraction G1 (10 g) was applied to ODS MPLC with 10 to 50% CH₃CN undergradient conditions and 550 mL fractions 11 to 17 were collected.Fraction 13 (410 mg) was chromatographed on SEPHADEX LH-20 with MeOHyielding 80 mg of yellow amorphous solid. This material was furtherpurified by successive Si gel PTLC chromatography withEtOAc-CHCl₃—MeOH—H₂O—HCOOH (65:25:10:0.8:0.1) (75 mg) followed byCHCl₃—MeOH—H₂O (8:2:1) (70 mg). Final purification by ODS preparativeHPLC with 60% MeOH gave 61 mg of compound 10 as a clear yellowglass-like solid.

Fraction 14 (1.5 g) was applied to SEPHADEX LH-20 and eluted with MeOHgiving 150 mL fractions 11 to 16. Fractions 13 to 16 (400 mg), 17 (300mg), and H2 to H4 (700 mg) were pooled and subjected to ODS MPLC withCH₃CN—MeOH—H₂O-TFA (20:20:60:0.1 to 40:40:20:0.1) under gradientconditions and 16 mL fractions K1 to K105 were collected. Fractions K22to K38 (430 mg) were combined and chromatographed on SEPHADEX LH-20 withMeOH giving fractions L1 to L2. Fraction L1 (300 mg) was applied to Sigel PTLC and developed twice with CHCl₃—MeOH—H₂O (8:2:0.1) giving asingle band that was further purified by ODS preparative HPLC with 60%MeOH to yield 31 mg of compound 11 as a clear glass-like solid.

Fractions L2 (130 mg) was applied to SEPHADEX LH-20 and eluted with MeOHto give 80 mg of a yellow amorphous solid. This material was dissolvedin MeOH and placed at −20° C. yielding 62 mg of precipitate. Theprecipitate was chromatographed twice by ODS preparative HPLC withCH₃CN—MeOH—H₂O-TFA (40:15:45:0.1) to give 30 mg of yellow solid. Furtherpurification (preparative HPLC) was achieved using 60 to 100% MeOH undergradient conditions yielding a single fraction that was reduced in vacuoand placed at −20° C. to providing 1 mg of compound 7 as a yellowpowder.

Fractions K50 to K55 were combined (98 mg) and subjected to SEPHADEXLH-20 chromatography with MeOH and 125 mL fractions M1 to M5 werecollected. Fraction M5 (40 mg) was dissolved in MeOH and left at roomtemperature whereupon 25 mg of compound 4 was obtained as fine yellowneedles.

Fractions K56 to K62 were pooled (130 mg) and applied to SEPHADEX LH-20with MeOH giving fractions N1 to N3. Fraction N1 (50 mg) was subjectedto further SEPHADEX LH-20 chromatography with MeOH giving a fraction (35mg) that was chromatographed using ODS preparative HPLC withCH₃CN—MeOH—H₂O-TFA (50:20:30:0.1). A single fraction was collected,reduced in vacuo, and placed at −20° C. yielding 6 mg of compound 5 asgolden yellow needles. Fraction N2 (7 mg) was further purified by ODSpreparative HPLC with CH₃CN—MeOH—H₂O-TFA (50:20:30:0.1) providing 1 mgof compound 12 as a yellow glass-like solid.

Fractions K63 to K77 were pooled and subjected to SEPHADEX LH-20 withMeOH and 100 mL fractions 01 to 05). Fraction 03 (30 mg) was applied toODS preparative HPLC with CH₃CN—MeOH—H₂O-TFA (50:20:30:0.1) yielding 6mg of yellow amorphous solid. This material was further purified by ODSpreparative HPLC under the same conditions and the resultant fractionreduced in vacuo and placed at −20° C. yielding 4 mg of compound 6 asfine yellow needles.

Fractions K94 to K100 were reduced in vacuo to dryness yielding 13 mg oforange amorphous solid. This material was dissolved in a minimal volumeof MeOH and left at −20° C. providing 7 mg of compound 3 as orangeneedles.

The 12 compounds were obtained in the yields shown in Table 1.

TABLE 1 Yield of the twelve compounds isolated from H. Fulva “Kwanzo”roots. Compound Yield (mg/kg) Compound Yield (mg/kg) 1 4.8 7 0.9 2 4.8 84.7 3 11.1 9 8.2 4 18.0 10 30.5 5 5.7 11 15.5 6 3.8 12 0.5

Table 2 shows the yields of the hexane, ethyl acetate, and methanolextracts from 2.0 kg of lyophilized roots and the yield of the compoundsin each of the extracts.

TABLE 2 Yield of compounds in extracts from 2.0 kg of lyophilized rootsYield ethyl hexane acetate methanol extract extract extract Compound (25g) (23 g) (130 g) Combined Yield 1 + 2 19 mg — —   19 mg 3 — 15.4 mg16.7 mg 22.1 mg 8 —  9.3 mg —  9.3 mg 4 —  1.0 mg 34.9 mg 35.9 mg 5 — —11.4 mg 11.4 mg 6 —  3.4 mg  4.1 mg  7.5 mg 1a + 2a NA NA NA NA 9 — —16.3 mg 16.3 mg 10 — —   31 mg   31 mg 11 — —   61 mg   61 mg 7 — —  1.8mg  1.8 mg

EXAMPLE 2

The physical characteristics of compounds 1 and 2 were determined to beas follows.

¹H NMR spectra were recorded at 500 and 600 MHz on Varian VRX (500 MHz)and Varian INOVA (600 MHz) instruments (Palo Alto, Calif.),respectively. ¹³C NMR spectra were obtained at 125 MHz on a Varian VRXinstrument. NMR spectra of compounds 1 and 2 were obtained in CDCl₃.Standard pulse sequences were employed for all 1D (¹H, ¹³C, DEPT,selective ¹H decoupling, and difference NOE) and 2D (DQF-COSY,long-range COSY, NOESY, HMQC, and HMBC) NMR experiments. Mass spectrawere acquired at the Michigan State University Mass SpectrometryFacility using a JOEL AX-505H double-focusing mass spectrometeroperating at 70 eV for EIMS analysis and a JOEL HX-110 double-focusingmass-spectrometer (Peabody, Mass.) operating in the positive ion modefor FABMS experiments. The UV spectra were recorded in EtOH using aShimadzu UV-260 recording spectrophotometer (Kyoto, Japan). IR spectrawere obtained on a Mattson Galaxy Series FTIR 3000 using WinFIRSTsoftware (Thermo Nicolet, Madison, Wis.). Optical rotations weremeasured with a Perkin-Elmer Polarimeter 341 (Shelton, Conn.).

Melting points were determined using a Thomas Model 40 Hot Stage(Philadelphia, Pa.).

The hexane extract was subjected to a series of chromatographicprocedures leading to the isolation of 12 mg of fine yellow needlesfollowing crystallization from CHCl₃-hexane. Initial inspection of the¹H and ¹³C NMR spectra of this product indicated a doubling of mostproton and carbon signals that suggested it was perhaps a large dimericcompound composed of more than 31 unique carbon nuclei. However,positive FABMS indicated a major signal at m/z 295 [M+H]⁺ that suggestedthe product was a mixture of two structurally related isomers each witha formula of C₁₈H₁₄O₄. This was supported by the presence a significantfragment ion at m/z 273 [M+H−H₂O]⁺. Further evidence was also providedby HMBC experiment that showed two sets of contours representing the2-3JCH connectivities for two compounds each composed of 18 carbon and14 proton spins. Extensive efforts to separate these two compoundsemploying Si gel MPLC and TLC, ODS MPLC and preparative HPLC, andcrystallization using a variety of solvent systems proved unsuccessful.Further attempts were made to separate the acetylated products (1a and2a) from one another, but this method also failed. Therefore, thestructure elucidation and full ¹H and ¹³C NMR assignments of compounds 1and 2 were performed on the inseparable 1:1 mixture of these twoconstitutional isomers.

Compounds 1 and 2 were determined to each be composed of substituted1-hydroxyanthraquinone moieties. Evidence for this came from acombination of HRFABMS with m/z 295.0971 [M+H]⁺ (calculated 295.0970)and spectroscopic studies. The IR spectrum of compound 1 and 2 exhibiteda number of diagnostic absorption bands at 3438 (broad, O—H stretch),1670 (C═O stretch, non-chelated), and 1633 cm⁻¹ (C═O stretch, chelated).The UV spectrum showed λ_(max)=403 nm suggesting the presence of asingle peri-hydroxyl functionality (Schripsema et al., Phytochem. 51:55-60 (1999)). This was supported by the ¹H NMR spectrum that revealedtwo sharp singlets at δ_(H) 12.90 and 12.95 that were both eliminatedupon D₂O exchange. Further evidence for the presence of a singlehydroxyl functionality in compounds 1 and 2 came from their acetylationproducts 1a and 2a that both exhibited the same molecular ion at m/z337.1068 [M+H]⁺ (calculated for C₂₀H₁₇O₅, 337.1076) representing theaddition of an acetyl moiety. The ¹H NMR spectrum of 1a and 2a no longerdisplayed any down field peaks between δ_(H) 12 and 13 while the ¹³C NMRspectrum exhibited new signals at δ_(C) 19.6 (—COCH₃) and 169.0(—COCH₃).

¹H NMR and DEPT experiments revealed the presence of two aromatic (δ_(C)20.2 q×2, 21.9 q, and 22.0 q) and one acetyl (δ_(C) 31.9 q×2) methylgroups in both compounds 1 and 2. Data from the HMBC experiment (Table3) provided evidence for the assignment of these functionalities asshown for compounds 1 and 2. Further support in favor of this conclusionwas obtained from long-range COSY and difference NOE experiments FIGS.2A and 2B. Both compounds 1 and 2 exhibited reciprocal NOE correlationsupon irradiation of the methyl protons of C-12 (both δ_(H) 2.59) and1-OH's (δ_(H) 12.95 and 12.90, respectively). In addition, NOEenhancements and long-range COSY correlations were noted between themethyl protons of C-13 (both δ_(H) 2.37) and the H-4 aromatic singlet(both δ_(H) 7.61). Together, these data confirmed the proposed ring Bassignments for compounds 1 and 2.

Compound 1 exhibited reciprocal NOE enhancements and COSY correlationsamongst H-7 (δ_(H) 7.58 d, J=7.5 Hz) and H-8 (δH 8.15 d, J=7.5 Hz), aswell as, between the methyl protons of C-14 (δH 2.51 s) and protons atpositions H-7 and H-5 (δH 8.04 s) (FIG. 2A). These evidence confirmedthat the aromatic methyl C-14 (δ_(H) 21.9) was attached at position 6 onring A of compound 1. Compound 2 differed by displaying reciprocal NOEenhancements and long-range COSY correlations between the methyl protonsof C-14 (δ_(H) 2.51 s) and protons H-6 (δ_(H) 7.58 d, J=7.5 Hz) and H-8(δ_(H) 8.05 s) (FIG. 2B). Similar NOE and COSY correlations were notedbetween H-6 and H-5 (δ_(H) 8.13 d, J=7.5 Hz). Therefore, the assignmentof the aromatic methyl C-14 (δ_(C) 22.0) was confirmed at position 7 onring A of compound 2. Both compounds 1 and 2 are newly discoveredcompounds which have been given the name kwanzoquinones A and B,respectively in honor of their biogenic source.

Kwanzoquinones A and B (compounds 1 and 2): yellow needles; meltingpoint 165-167° C.; UV λ_(max) (EtOH) 212, 262, 287, 403 nm; IR (KBr)υ_(max) 3438, 1700, 1696, 1691, 1685, 1670, 1652, 1630, 1595, 1559 cm⁻¹;¹H NMR ¹³C NMR data, see Table 3; HRFABMS m/z 295.0971 [M+H]⁺(calculatedfor C₁₈H₁₅O₄, 295.0970).

TABLE 3 NMR Spectra Data for Kwanzoquinones A (1) and B (2) in CDCL₃^(a) 1 2 position δ_(n)(J in Hz)^(b) δ_(c) ^(c) HMBC^(d) δ_(n)(J inHz)^(b) δ_(c) ^(c) HMBC^(d)  1 159.6(s) 1-OH 159.6(s) 1-OH  2114.4^(e)(s) 1-OH, H- 114.5^(e)(s) 1-OH, H- 13, H-4 13, H-4  3144.7^(f)(s) H-4, H-13 144.9^(f)(s) H-4, H-13  4  7.61(s) 121.5(d) H-13 7.61(s) 121.5(d) H-13  4a 133.1(s) H-4 133.19(s) H-4  5  8.04(s)127.8(d) H-14  8.13(d, 7.5) 127.7(d) H-6  6 146.2(s) H-8, H-14  7.58(d,7.5) 135.6(d) H-8, H-14  7  7.58(d, 7.5) 135.1(d) H-5, H-14 145.6(s)H-5, H-14  8  8.15(d, 7.5) 127.1(d) H-7  8.05(s) 127.2(d) H-14  8a133.4(s) H-8 131.2(s) H-8  9 188.1(s) H-8 188.5(s) H-8  9a 135.7^(g)(s)1-OH, H-4 135.8^(g)(s) 1-OH, H-4 10 182.3(s) H-4, H-5 181.9(s) H-4, H-510a 130.9(s) H-5 133.0(s) H-5 11 203.0(s) H-12 203.0(s) H-12 12  2.59(s) 31.9(q)  2.59(s)  31.9(q) 13  2.37(s)  20.2(q) H-4  2.37(s)  20.2(q)H-4 14  2.51(s)  21.9^(h)(q) H-5, H-7  2.51(s)  22.0^(h)(q) H-6, H-81-OH 12.95(s) 12.90(s) ^(a)All spectra were recorded using 12 mg of a1:1 mixture of compounds 1 and 2 dissolved in 1 mL of CDCL₃ with a 5 mmprobe at 25° C. ^(b)Recorded at 500 MHz. ^(c)Recorded at 125 MHz.Multiplicities were determined by DEPT experiment. ^(d)HMBC data wererecorded using a ^(n)J_(CH) = 8 Hz and are expressed as protonsexhibiting ²⁻³J_(CH) couplings to the carbons as indicated.^(e-h)Assignments may be interchanged.

EXAMPLE 3

The physical characteristics for compound 3 were determined as inExample 2 except that all NMR spectra were recorded in DMSO-d₆(Cambridge Isotope Laboratories, Inc., Andover, Mass.). Thecharacteristics are as follows.

The MeOH extract was subjected to repeated ODS and SEPHADEX LH-20 gelcolumn chromatography yielding compounds 3-12. Following purification,compound 3 was obtained from MeOH as orange needles. HREIMS (m/z270.0532 [M]⁺ (calculated for C₁₅H₁₀O₅, 270.0528)) and spectral evidence(IR, UV, ID and 2D NMR) confirmed that compound 3 (1,2,8-trihydroxy3-methylanthraquinone) had been previously isolated from Myrsineafricana L. (Myrsinaceae) and was given the trivial name2-hydroxychrysophanol (Li and McLaughlin, J. Nat. Prod. 52: 660-662(1989); Midiwo and Arot, Int. J. BioChemiPhysics 2: 115-116 (1993)).Previous studies had only given partial ¹H and no ¹³C NMR assignmentsfor this compound; therefore, we undertook a thorough NMR investigationof compound 3 in order to confirm its proposed structure. This is thefirst report of compound 3 from daylilies and the first report of itscomplete ¹³C NMR spectral date (Table 4).

2-Hydroxychrysophanol (compound 3): orange needles; melting point239-240° C.; UV λ_(max) (EtOH) (loge) 208 (4.19), 235 (4.05), 258(4.11), 426 (3.73) nm; IR (KBr) υ_(max) 3408, 1653, 1620, 1560, 1473,1456, 1434, 1310, 1271, 1190, 1023 cm⁻¹; ¹H NMR (DMSO-d₆) δ_(H) 12.04(1H, brs, 1-OH), 11.90 (1H, s, 8-OH), 10.34 (1H, brs, 2-OH), 7.76 (1H,dd, 8.0, 7.5, H-6), 7.66 (1H, dd, 7.5, 1.0, H-5), 7.55 (1H, s, H-4),7.31 (1H, dd, 8.0, 1.0, H-7), 2.26 (1H, s, 3-CH₃; ¹³C NMR, see Table 4;EIMS m/z 270 [M]⁺ (100), 253(2), 242(8), 213(4), 196(3), 185(2), 168(5),139(11); HREIMS m/z 270.0532[M]⁺ (calculated for C₁₅H₁₀O₅, 270.0528)(for literature values refer to Li and McLaughlin, ibid.; Midiwo andArot, ibid.).

TABLE 4 ¹³C NMR Assignments for Compounds 3 to 7 and 9^(a) position 3 45 6 7 9  1 194.4s 153.9s 153.9s 149.1s 153.4s 158.6s  2 150.2s 147.7s147.7s 148.4s 146.0s 131.2s  3 132.3s 141.4s 141.5s 136.7s 145.3s 140.4s 4 122.8d 121.5d 121.4d 119.0d 117.9d 112.0d  4a 123.1s 128.02 128.0s123.3s 128.2s 136.2s  5 119.0d 119.0d 119.0d 119.1d 119.1d 118.1d  6137.3d 137.1d 137.1d 137.4d 137.2d 135.9d  7 123.7d 124.1d 124.0d 123.7d124.0d 124.2d  8 161.2s 161.2s 161.2s 161.3s 161.2s 161.3s  8a 115.9s115.9s 115.9s 116.0s 116.1s 116.7s  9 192.2s 191.5s 191.4s 192.3s 191.7s189.2s  9a 114.3s 115.2s 115.2s 114.6s 115.7s 122.4s 10 180.1s 180.8s180.6s 180.2s 180.8s 181.8s 10a 133.7s 133.2s 133.1s 133.8s 133.3s132.3s 11 16.4q 17.2q 17.2q 57.8t 58.1t 19.5q 12 167.8s  1′ 102.9d102.8d 102.7d  2′ 74.2d 74.0d 74.1d  3′ 76.3d 76.0d 76.2d  4′ 69.7d69.7d 69.7d  5′ 77.3d 73.8d 77.2d  6′ 60.8t 63.7t 60.7t  1″ 166.4s  2″41.1t  3″ 167.4s ^(a)Data recorded in DMSO-d₆ at 125 MHz at 25° C.Multiplicities were determined by DEPT experiment and confirmed byanalysis of HMQC spectra.

EXAMPLE 4

The physical characteristics for compound 4 were determined as inExample 3. The characteristics are as follows.

The MeOH extract was subjected to repeated ODS and SEPHADEX LH-20 gelcolumn chromatography yielding compounds 4. Compound 4 was obtained asyellow needles and exhibited many spectral characteristics similar to 3.The IR spectrum of 4 revealed absorption bands at 3455 (broad, O—Hstretch), 1671 (C═O stretch, non-chelated), and 1624 cm⁻¹ (C═O stretch,chelated). The UV spectrum presented a λ_(max)=429 nm that was in accordwith the presence of two peri-hydroxyl functionalities (Schripsemaibid.; Brauers et al., J. Nat. prod. 63: 739-745 (2000); Li et al., J.Nat. Prod. 63: 653-656 (2000)). In addition, the ¹H NMR spectrum showedtwo down field peaks (δ_(H) 12.00 s and 12.04 brs) that wereexchangeable with D₂0. Together this evidence supported the presence ofa 1,8-dihydroxyanthraquinone chromaphore for compound 4.

FABMS gave m/z 433 [M+H]⁺ that represented a molecular composition ofC₂₁H₂₁O₁₀. ¹H NMR provided important evidence for the substitutionpattern of rings B and A in compound 4. Three protons representing an.ABC spin system at δ_(H) 7.70 (dd, J=1.0, 7.5 Hz), 7.79 (dd, J=7.5, 8.0Hz), and 7.36 (dd, J=1.0, 8.0 Hz) were identified as occupy contiguouspositions attached to C-5, C-6, and C-7, respectively on ring A ofcompound 4. ¹³C NMR and DEPT experiments (Table 4) provided furtherevidence for the identity of the substituents attached to ring B ofcompound 4 with one methine (δ_(C) 121.5), one C-linked (δ_(C) 141.4)methyl (δ_(C) 17.2), and two quaternary carbon (δ_(C) 147.7 and 153.9)linked with a hetero-atom. These carbons were assigned positions in ringB of compound 4 based on their respective chemical shifts and theresults from HMBC and HMQC experiments. Five additional methine (δ_(C)69.7, 74.2, 76.3, 77.3, and 102.9) and one methylene (δ_(C) 60.8) spinswere observed that exhibited chemical shift values that coincided withthose for a glucopyranose moiety. The glucopyranose was assigned aβ-configuration based on the coupling of H-1′ (δ_(H) 5.07, d, J=7.5 Hz).The complete structure of compound 4 was confirmed based on HMBCexperiment. Compound 4 is a newly discovered anthraquinone glycosidewhich has been given the name kwanzoquinone C.

Kwanzoquinone C (compound 4): fine yellow needles; melting point233-234° C.; [α]²⁰ _(D)−46° (c 0.031, EtOH); UV λ_(max) (EtOH) (logε)206(4.20), 227(4.23), 260(4.17), 429(3.78) nm; IR (KBr) υ_(max) 3422,1671, 1624, 1559, 1473, 1382, 1373, 1293, 1263, 1067 cm⁻¹; ¹H NMR(DMSO-d₆) δ_(H) 12.04 (1H, brs, 8-OH), 12.00 (1H, s, 1-OH), 7.79 (1H,dd, J=7.5, 8.0 Hz, H-6), 7.70 (1H, dd, J=1.0, 7.5 Hz, H-5), 7.61 (1H, s,H-4), 7.36 (1H, dd, J=1.0, 8.0 Hz), 5.07 (1H, d, J=7.5 Hz, H-1′), 3.60(1H, ddd, J=2.0, 5.5, 12.0 Hz, H-6a′), 342 (1H, ddd, J=6.0, 11.5, 11.5Hz, H-6b′), 3.31 (1H, m, H-2′), 2.35 (1H, m, H-3′), 3.16 (1H, m, H-4′),3.13 (1H, m, H-5′), 2.42 (3H, s, H-11); ¹³C NMR data, see Table 4;HRFABMS m/z 433.1139 [M+H]⁺ (calculated for C₂₁H₂₁O₁₀, 433.1135).

EXAMPLE 5

The physical characteristics for compound 5 were determined as inExample 3. The characteristics are as follows.

The MeOH extract was subjected to repeated ODS and SEPHADEX LH-20 gelcolumn chromatography yielding compounds 5. The molecular formula ofcompound 5 was determined to be C₂₄H₂₂O₁₃ based on FABMS analysis thatexhibited m/z 519 [M+H]⁺. The spectral data of compound 5 were verysimilar to those obtained for compound 4. The most significantdifference was observed in the ¹H and ¹³C NMR spectra (Table 4) with theaddition of three new carbon signals at δ_(C) 41.1, 166.4, and 167.4 anda new proton resonance at δ_(H) 3.23 integrating for two hydrogens.These chemical shifts were characteristic of those expected for amalonyl moiety. The linkage of the malonyl group in compound 5 wasestablished as malonyl-(1-6)-β-D-glucopyranoside based on the observeddown field shift of C-6′ to δ_(C) 63.7 verses that observed for compound4 (Δ=+2.9 ppm). These observations were verified by HMBC analyses (FIG.3) which exhibited weak ⁴J_(CH) correlations from H-6a′ (δ_(H) 4.12) andH-6b′ (δ_(H) 4.27) to C-1″ (δ_(C) 41.1) and H-2″ (δ_(H) 3.23) to C-6′(δ_(C) 63.7). This confirmed compound 5 was a new anthraquinonemalonyl-glucoside which was then named kwanzoquinone D.

Kwanzoquinone D (compound 5): golden-yellow needles; melting point174-175° C.; [α]²⁰ _(D)−313′ (c 0.008, EtOH); UV λ_(max) (EtOH) (logε)205(4.28), 227(4.35, 260(4.31), 290 sh (3.91), 430(3.96) nm; IR (KBr)υ_(max) 3430, 1434, 1717, 1699, 1670, 1653, 1559, 1457, 1268, 1066 cm⁻¹;¹H NMR (DMSO-d₆) δ_(H) 12.57 (1H, brs, 1-OH), 11.96 (1H, s, 8-OH), 7.77(1H, dd, J=7.5, 8.0 Hz, H-6), 7.67 (1H, dd, J=1.0, 7.5 Hz, H-5), 7.57(1H, s, H-4), 7.33 (1H, dd, J=1.0, 8.0 Hz), 5.06 (1H, d, J=7.5 Hz,H-1′), 4.27 (1H, dd, J=2.5, 11.9 Hz, H-6a′), 4.12 (1H, dd, J=6.5, 11.9Hz, H-6b′), 3.38 (1H, m, H-5′), 3.33 (1H, m, H-2′), 3.28 (1H, m, H-3′),3.23 (2H, s, H-2″), 3.21 (1H, m, H4′), 2.37 (3H, s, H-11); ¹³C NMR data,see Table 4; HRFABMS m/z 519.1139 [M+H]⁺ (calculated for C₂₄H₂₃O₁₃,59.1151).

EXAMPLE 6

The physical characteristics for compound 6 were determined as inExample 3. The characteristics are as follows.

The MeOH extract was subjected to repeated ODS and SEPHADEX LH-20 gelcolumn chromatography yielding compound 6. EIMS analysis of compound 6gave a molecular ion of m/z 286 [M]⁺ indicating a molecular formula ofC₁₅H₁₀O₆. The UV (λ=426 nm) and IR (absorption bands at 3469 (broad, O—Hstretch), 1667 (C═O stretch, non-chelated), and 1620 cm⁻¹ (C═O stretch,chelated)) spectra suggested a 1,8-dihydroxyanthraquinone chromaphorefor compound 6. The ¹H NMR spectrum provided evidence for fourexchangeable protons at δ 12.06, 11.92, 10.47, and 5.40 representingthree aromatic and one aliphatic hydroxyl functionalities. An ABC spinsystem was observed with protons at 5H 7.70 (dd, J=0.5, 7.8 Hz), 7.78(overlapping dd, J=7.8, 7.8 Hz), and 7.33 (dd, J=0.5, 7.8 Hz) occupycontiguous positions attached to C-5, C-6, and C-7, respectively on ringA of compound 6.

¹H and ¹³C NMR and DEPT experiments of compound 6 (Table 4) gaveevidence that ring A possessed quaternary carbons with ortho-hydroxylfunctionalities (δ 149.1 s and 148.4 s), a hydroxy-methylene moietyδ_(H) 4.59 s, 2H and δ_(C) 57.8 t) attached to a quaternary carbon (6c136.7), and a methine (6c 119.0). An HMBC experiment was used to makefull assignments of these proton and carbon spins as shown for compound6 (FIG. 4). Compound 6 is a newly discovered which has been namedkwanzoquinone E.

Kwanzoquinone E (compound 6): fine yellow needles; melting point196-197° C.; UV λ_(max) (EtOH) (loge) 209(4.32), 235(4.10), 258(4.27),354(3.72), 426(3.76) nm; IR (KBr) υ_(max) 3469, 1652, 1619, 1559, 1473,1458, 1382, 1321, 1273, 1092 cm⁻¹; ¹H NMR (DMSO-d₆) δ_(H) 12.06 (1H,brs, 1-OH), 11.92 (1H, s, 8-OH), 10.47 (1H, brs, 2-OH), 7.87 (1H, d,J=0.5 Hz, H-4), 7.78 (1H, dd, J=7.8, 7.8 Hz, H-6), 7.70 (1H, dd, J=0.5,7.8 Hz, H-5), 7.33 (1H, dd, J=0.5, 7.8 Hz, H-7, 5.40 (1H, brs, 11-OH),4.59 (2H, s, H-11); ¹³C NMR data, see Table 4; EIMS m/z 286 [α]⁺ (62),268(89), 240(56), 212(100), 184(50), 155(14), 128(19), 120(19); HREIMSm/z 286.0479 [α]⁺(calculated for C₁₅H₁₀O₅, 286.0477).

EXAMPLE 7

The physical characteristics for compound 7 were determined as inExample 3. The characteristics are as follows.

The MeOH extract was subjected to repeated ODS and SEPHADEX LH-20 gelcolumn chromatography yielding compounds 7. Compound 7 exhibitedspectral data similar to 6 with the addition of five methine (δ_(C)69.7, 74.1, 76.2, 77.2, and 102.7) and one methylene (δ_(C) 60.7) spinsthat exhibited chemical shift values that coincided with those for aglucopyranose moiety. The addition of a glucopyranose moiety wasconfirmed by HRFABMS which gave m/z 449.1082 [M+H]⁺ (calculated 449.1084for C₂₁H₂₀O₁₁) representing a molecular formula of C₂₁H₂₀O₁₁ forcompound 7. The glucopyranose moiety was determined to be O-linked atposition 11 due to the down field shift of this carbon signal to δ_(C)58.1 (Δ=+0.4) and the change in the splitting pattern of the attachedprotons. While the enantiotopic C-1 protons of compound 6 (δ_(H) 4.59,2H) appeared as a singlet, the diastereotopic C-11 protons of compound 7(6H 4.65, 1H and 4.73, 1H) were each a doublet (J=16.0 Hz) in achiralsolvent (0.75 mL DMSO-d₆ with 2 drops D₂O). The assignments of allproton and carbon (Table 4) spins in compound 7 were confirmed by HMBCexperiment. Compound 7 is a newly discovered conjugated anthraquinoneglucoside which has been given the name kwanzoquinone F.

Kwanzoquinone F (compound 7): yellow powder; melting point 204-206° C.;[α]²⁰ _(D)−38° (c 0.01, EtOH); UV λ_(max) (EtOH) (logε) 228(4.04),259(4.03), 291(3.57), 432(3.68) nm; IR (KBr) υ_(max) 3450, 1698, 1684,1652, 1635, 1559, 1540, 1457, 1262, 1027 cm⁻¹; ¹H NMR (0.75 mL DMSO-d₆/2drops D₂O) δ_(H) 7.88 (1H, s, H-4′), 7.79 (1H, dd, J=7.5, 8.0 Hz, H-6),7.71 (1H, dd, J=1.0, 7.5 Hz, H-5), 7.36 (1H, dd, J=1.0, 8.0 Hz, H-7),5.07 (1H, d, J=7.5, H-1′), 4.37 (1H, d, J=16.0 Hz, H-11a), 4.65 (1H, d,J=16.0 Hz, H-11b), 3.60 (1H, d, J=3.0, 12.5 Hz, H-6a′), 3.40 (1H, dd,J=5.5, 12.0 Hz, H6-b′), 3.26 (1H, m, H-2′), 3.25 (1H, m, H-3′), 3.15(1H, m, H-4′), 3.12 (1H, m, H-5′); ¹³C NMR data, see Table 4; HRFABMSm/z 433.1132 [M+H]+(calculated for C₂₁H₂₁O₁₀, 433.1135).

EXAMPLE 8

The MeOH extract was subjected to repeated ODS and SEPHADEX LH-20 gelcolumn chromatography yielding compounds 8. Compound 8 was obtained asan amorphous yellow powder and its spectral data were found to matchthose reported for the known anthraquinone rhein (Danielsen and Aksnes,Magn. Reson. Chem. 30: 359-360 (1992)).

EXAMPLE 9

The physical characteristics for compound 9 were determined as inExample 3. The characteristics are as follows.

The MeOH extract was subjected to repeated ODS and SEPHADEX LH-20 gelcolumn chromatography yielding compounds 9. Compound 9 exhibitedspectral-data that were similar to compound 8 with the main differencesin the ¹H NMR spectrum being the loss of an aromatic doublet (ca δ_(H)8.15, 1H, J=1.5 Hz) and the concomitant loss of splitting in the protonsignal at δ_(H) 7.59 (s, 1H) indicating that position 2 in ring B ofcompound 9 was substituted. These observations coincided with theappearance of an aromatic methyl (δ_(H) 2.67 s, 3H and δ_(C) 19.5 q) andthe down field shift of C-2 in compound 8 from δ_(C) 124.2 to 131.2(Δ=+7.0 ppm) in compound 9 (Table 4). HMBC experiment was able toconfirm that this methyl was a substituent of C-2 based on thelong-range coupling of the C-11 methyl protons to C-2 (δ_(C) 131.2) andC-3 (δ_(C) 140.4). Compound 9 is a newly discovered anthraquinone whichhas been named kwanzoquinone G.

Kwanzoquinone G (compound 9): yellow powder; melting point 235-236° C.;λ_(max) (EtOH) (logε) 219(4.25), 283(4.19), 413(3.63) nm; IR (KBr)υ_(max) 3420, 1717, 1700, 1670, 1634, 1577, 1365, 1320, 1261, 1223 cm⁻¹;¹H NMR (DMSO-d₆) δ_(H) 12.82 (1H, s, 8-OH), 12.81 (2H, brs, 1-OH and12-OH), 7.67 (1H, dd, J=8.1, 8.1 Hz, H-6), 7.57 (1H, dd, J=1.2, 8.1 Hz,H-5), 7.56 (1H, s, H-4), 7.28 (1H, dd, J=1.5, 8.1 Hz, H-7), 2.67 (3H, s,H-1); ¹³C NMR data, see Table 4; HRFABMS m/z [299.0547 M+H]+(calculatedfor C₁₆H₁₁O₆, 299.0556).

EXAMPLE 10

The physical characteristics for compound 10 were determined as inExample 3. The characteristics are as follows.

The MeOH extract was subjected to repeated ODS and SEPHADEX LH-20 gelcolumn chromatography yielding compounds 3-12. FABMS of compound 10provided a molecular ion of m/z 525 [M+H]⁺ and the ¹³C NMR spectrumexhibited ten sp² carbon signals between 110 and 155 ppm along with 12additional sp³ carbon signals that were characteristic of a rutinosemoiety. In light of the presence of three additional carbon signals thatrepresented an aromatic methyl (δ_(C) 19.0) and an acetyl moiety (δ_(C)31.9 and 204.4), it was determined that compound 10 was a substitutednaphthalene diglycoside. HMQC and HMBC experiments established theaglycone portion of compound 10 as2-acetyl-3-methyl-1,8-dihydroxynaphthalene, dianellidin. Furtherscrutiny of the HMBC data provided for the assignment of an 8-O-linkageto the rutinoside moiety based on a correlation from H-1′ (δ_(H) 5.04,d, J=7.5 Hz) to C-8 (δ_(c) 154.2 s). According to these data, compound10 was identified as dianellin, previously isolated from Dianella spp.(Liliaceæ) (Batterham et al., Aust. J. Chem. 14: 637-642 (1961)). Thisis the first report showing compound 10 is present in daylilies and thefirst report detailing its ¹H and ¹³C NMR spectral data.

Dianellin (compound 10): white needles; melting point 156-157° C.; [α]²⁰_(D)−137′ (c 0.01, EtOH); UV λ_(max) (EtOH) (loge) 225(4.75), 301(3.80),334(3.78) nm; IR (KBr) υ_(max) 3416, 2923, 1651, 1633, 1579, 1467, 1443,1356, 1270, 1067 cm⁻¹; ¹H NMR (DMSO-d₆) δ_(H) 9.53 (1H, brs, 1-OH), 7.47(1H, dd, J=1.0, 8.0 Hz, H-5, 7.40 (1H, dd, J=8.0, 8.0 Hz, H-6), 7.30(1H, dd, J=1.0, 8.0 Hz, H-7), 7.21 (1H, s, H-4, 5.04 (1H, d, J=7.5 Hz,H-1′), 4.62 (1H, d, J=1.5 Hz, H-1″), 3.93 (1H, dd, J=1.5, 11.0 Hz,H-6a′), 3.68 (1H, m, H-2″), 3.59 (1H, m, H-5′), 3.50 (2H, m, H-4′), 3.18(1H, m, H-4′), 2.52 (3H, s, H-12), 2.25 (3H, s, H-13), 1.12 (3H, d, J=6Hz, H-6′); ¹³C NMR (DMSO-d₆) δ_(C) 204.4 (s, C-11), 154.2 (s, C-8),150.2 (s, C-1), 135.7 (s, C-10), 132.8 (s, C-3), 127.3 (d, C-6), 125.2(s, C-2), 122.3 (d, C-5), 119.4 (d, C-4), 113.2 (s, C-9), 110.7 (d,C-7), 102.6 (d, C-1′), 100.7 (d, C-1″), 76.2 (d, C-3′), 76.0 (d, C-5′),73.3 (d, C-2′), 71.9 (d, C-4″), 70.7 (d, C-3″), 70.4 (d, C-2″), 70.1 (d,C-4′), 68.4 (d, C-5″), 66.6 (t, C-6′), 31.9 (q, C-12), 19.0 (q, C-13),17.7 (q, C-6″); HRFABMS m/z 525.1970 [M+H]⁺ (calculated for C₂₅H₃₃O₁₂,525.1972)

EXAMPLE 11

The physical characteristics for compound 11 were determined as inExample 3. The characteristics are as follows.

The MeOH extract was subjected to repeated ODS and SEPHADEX LH-20 gelcolumn chromatography yielding compounds 11. The ¹H and ¹³C NMR and DEPTdata of compound 11 were very similar to those observed for compound 10with the loss of one aromatic methine spin that was replaced by aquaternary carbon (5c 148.3) that was linked to a hetero-atom. FABMSanalysis yielded a molecular ion of m/z 541 [M+H]⁺ which accounted forthe addition of an oxygen atom giving a molecular formula of C₂₅H₃₂O₁₃.A comparison of the ¹H and ¹³C NMR data for the aglycone portion ofcompound 11 with that previously reported for the naphthalene glycosidestelladerol, demonstrated that both possessed the same aglycon moiety.These compounds differed, however, with respect to their glycosideportion. HMBC correlation data for compound 11 (FIG. 5) showed that itpossessed an 8-O-β-D-rhamnopyranosyl-(1→6)-β-D-glucopyranoside moiety.Significant HMBC correlations that were used to deduce theseconnectivities included those observed for H-1′ (δ_(H) 4.87, d, J=7.5Hz) to C-8 (δ_(C) 146.7 s), and H-6a′ (δ_(H) 3.92 m) and H-6b′ (δ_(H)3.52 m) to C-1″ (δ_(C) 100.7 d), as well as, from H-1″ (δ_(4.61) m) toc-6′ (δ_(C) 66.6 t). Based on these data, compound 11,5-hydroxydianellin(1-(1,5,8-trihydroxy-3-methyl-napthalen-2-yl)-ethanone-8-O-β-D-rhamnopyranosyl-(1→6)-β-D-glucopyranoside),was identified as a newly discovered naphthalene glycoside.

5-Hydroxydianellin (compound 11): yellow amorphous solid; melting point152-153° C.; [α]²⁰ _(D)−212° (c 0.01, EtOH); UV λ_(max) (EtOH) (logε)224(4.92), 318(4.13), 346(4.15) nm; IR (KBr) υ_(max) 3420, 1698, 1684,1653, 1635, 1559, 1457, 1364, 1257, 1059 cm⁻¹; ¹H NMR (DMSO-d₆) δ_(H)9.71 (2H, brs, 1-OH and 5-OH), 7.43 (1H, s, H-4), 7.16 (1H, d, J=8.0,H-7), 6.76 (1H, d, J=8.0, H-6), 4.87 (1H, d, J=7.5, H-1′), 4.61 (1H, m,H-1″), 3.92 (1H, m, H-6a′), 3.69 (1H, brs, H-2″), 3.52 (1H, m, H-6b′),3.51 (1H, m, H-5′), 3.50 (1H, m, H-3″), 3.48 (1H, H-5″), 3.34 (2H, m,H-2′ and H-3″), 3.21 (1H, m, H-4″, 3.18 (1H, m, H-4′), 2.51 (3H, s,H-12), 2.26 (3H, s, H-13), 1.14 (3H, d, J=6 Hz, H-6″); ¹³C NMR (DMSO-d₆)δ_(C) 204.7 (s, C-11), 150.2 (s, C-1), 148.3 (s, C-5), 146.7 (s, C-8),131.3 (s, C-3), 126.4 (s, C-10), 125.6 (s, C-2), 114.2 (s, C-9), 113.8(d, C-4), 111.9 (d, C-7), 108.6 (d, C-6), 103.5 (d, C-1′), 100.7 (d,C-1″), 76.3 (d, C-3′), 75.9 (d, C-5′), 73.3 (d, C-2′), 72.0 (d, C-4″),70.8 (d, C-3″), 70.5 (d, C-2″), 70.0 (d, C-4″), 68.4 (d, C-5″), 66.6 (t,C-6′), 31.9 (q, C-12), 19.3 (q, C-13, 17.7 (q, C-6″); HRFABMS m/z541.1910 [M+H]⁺ (calculated for C₂₅H₃₃O₁₃, 541.1921).

EXAMPLE 12

The physical characteristics for compound 12 were determined as inExample 3. The characteristics are as follows.

The MeOH extract was subjected to repeated ODS and SEPHADEX LH-20 gelcolumn chromatography yielding compounds 12. Compound 12 was obtained asa clear glass-like solid and identified as5,7,3,4-tetrahydroxy-6-methylflavone (6-methylluteolin) that waspreviously reported from Salvia nemorosa L. (Lammiaceæ) (Milovanovic etal., J. Serb. Chem. Soc. 61: 423-429 (1996). Its structure was confirmedbased on through 1D and 2D NMR studies and by comparisons of its UV andIR spectral data with those reported in the prior art. This is the firstreport showing compound 12 is present in daylilies and the first reportdetailing its ¹H and ¹³C NMR spectral data.

6-Methyllueolin (compound 12): yellow glass-like solid; UV and IR datawere identical to values in Milovanovic et al. (ibid.); ¹H NMR (DMSO-d₆)δ_(H) 10.92 (1H, s, 5-OH), 9.71 (1H, s, 7-OH), 9.55 (1H, s, 4′-OH), 9.23(1H, s, 3′-OH), 7.40 (1H, d, J=2.0 Hz, H-2′), 7.16 (1H, dd, J=2.0, 8.5Hz, H-6′), 6.80 (1H, d, J=8.5 Hz, H-5′), 6.47 (1H, s, H-3), 6.32 (1H, s,H-8), 1.92 (3H, s, —CH₃); ¹³C NMR (DMSO-d₆) δ_(c) 180.1 (s, C-4), 165.0(s, C-5), 164.2 (s, C-9), 154.5 (s, C-7), 147.4 (s, C-4′), 145.6 (s,C-3′), 145.3 (s, C-2), 123.9 (d, C-6′), 123.5 (s, C-1′), 117.5 (d,C-2′), 115.8 (d, C-5′), 109.8 (d, C-3), 105.8 (s, C-6), 102.8 (s, C-10),90.2 (d, C-8), 7.5 (q, —CH₃); HRFABMS m/z 301.0709 [M+H]⁺ (calculatedfor C₁₆H₁₃O₆, 301.0712).

EXAMPLE 13

Compounds 1 to 11, including compounds 1a and 2a, were assayed forinhibitory activity against multiple life-stages (cercariae,schistosomula, and adult) of the human pathogenic trematode Schistosomamansoni. Anthraquinones assayed for toxicity on Schistosome cercariae.The results show that compounds 3 and 6 were inhibitory. The assays wereperformed as follows.

Schistosoma mansoni (Puerto Rican strain) cercariae were collected frominfected Biomphalaria glabrata snails by light induction as taught bySalter et al. in J. Biol. Chem. 275: 38667-38673 (2000).

One mg of each compound was separately dissolved in 100 μL DMSO. To thissolution, 19.9 mL distilled water was added to make a 1:200 dilution ofthe solution to produce a stock solution containing 50 μg/mL of thecompound.

For testing the inhibitory effect of each compound, 100 μL of stocksolution containing the compound at 50 μg/mL was added to 100 μL freshlyshed S. mansoni cercariae (approx. 50) in the wells of a 96-well assayplate to give a final volume of 200 μL wherein the concentration of thecompound was 25 μg/mL. The mobility and motility of the cercariae wasmonitored over time.

At a concentration of 25 μg/mL, compound 3 immobilized the cercariaewhich sank to the bottom of the wells within 10 to 15 seconds after theaddition of the solution containing compound 3. After 2, 5, 10, 15 and30 minutes of exposure to compound 3, the solution was removed from thewells and replaced with 200 μL of fresh water. After 16 hours, 50% ofcercariae which had been exposed to compound 3 for each of the exposuretimes were still alive and fairly active. The guts of the live cercariaewere dark (a phenomenon not seen in the control wells). After 24 hours,the mortality remained at 50%. Therefore, the length of time thecercariae were exposed to compound 3 had no significant effect onsurvival of the cercariae. Even when compound 3 was diluted to 3.25μg/mL, the cercariae were immobilized after 45 minutes exposure.

At a concentration of 25 μg/mL, compound 6 immobilized the cercariaewhich sank to the bottom of the wells within 12 to 14 minutes after theaddition of the solution containing compound 6. After 2, 5, 10, 15 and30 minutes of exposure to compound 6, the solution was removed from thewells and replaced with 200 μL of fresh water. After 16 hours, 25 to 30%of cercariae which had been treated with compound 6 for each of theexposure times were still alive but not active. The guts of the livecercariae were dark here as well. After 24 hours, all the cercariaewhich had been treated with compound 6 for each of the exposure timeswere dead (100% mortality). Therefore, the length of time the cercariaewere exposed to compound 6 had no significant effect on survival of thecercariae.

None of the other compounds, including the glycosides of compounds 3 and6 (compounds 4 and 7, respectively) exhibited any inhibitory activityagainst S. mansoni.

The results show that compound 3 is more potent at immobilizingcercariae but less toxic or lethal than compound 6 in longer-termfollow-up. In light of the results, a regimen for treating a patientinfected with a human pathogenic trematode would include providing acomposition comprising both compound 3 to rapidly immobilize thecercariae and compound 6 to kill all the cercariae. At 0.25 ppm,compounds 3 and 6 produced essentially 100% mortality of cercariae overtime as shown in FIG. 2.

Because compounds 4 and 5 can be hydrolyzed in the gut to compound 3 andcompound 7 can be hydrolyzed in the gut to compound 6, a regimen fortreating a patient infected with a human pathogenic trematode wouldinclude providing a composition containing compound 7 and at least onecompound selected from the group consisting of compound 4 and compound5. Further regimens would include compositions comprising compound 3 andcompound 6 and compound 7 and at least one compound selected from thegroup consisting of compound 4 and compound 5.

EXAMPLE 14

Compounds 1-11, including 1a and 2a, were tested for activity againstmultiple life stages (cercariae, schistosomula, and adult) of the humanpathogenic trematode Schistosoma mansoni.

The cercariae assays were performed as follows. S. mansoni (Puerto Ricanstrain) cercariae were obtained from infected Biomphalaria glabratasnails by light induction. Details regarding the methods used for themaintenance of both S. mansoni and B. glabrata cultures was as describedin Salter et al., J. Biol. Chem. 275: 38667-38673 (2000). A total of50-100 cercariae in 100 μL distilled water were collected and placed inCOSTAR 96-well vinyl assay plates (COSTAR Corp., Acton, Mass.). Stocksolutions of compounds 1-11, including 1a and 2a, were prepared bydissolving 1 mg of test compound in 100 μL of DMASO and 19.9 mL ofdistilled water. The stock solution was further diluted as needed and100 μL aliquots were added to each well. Cercariae mobility (that is,tail movement and swimming behavior) was observed under a dissectingmicroscope. Viability of the cercariae was determined by removing thetest compounds after about ten hours and replacing it with fresh water.Recovery from exposure to the test compounds was assessed after 24hours.

The schistosomula assays were performed as follows. Schistosomula wereprepared from S. mansoni cercariae by shearing the tails and incubatingthe organisms for two days in RPMI-1640 media containing penicillin andstreptomycin and fetal bovine serum in flat-bottomed COSTAR 96-well CELLCULTURE CLUSTER tissue culture plates. Test compounds prepared as abovewere added to the media and the schistosomula were observed for changesin movement, feeding, and viability.

The adult assays were performed as follows Adult worms were perfusedfrom Syrian Golden hamsters as described in Davies et al., Science 294:1358-1361 (2001). Twenty male and female adult worms were cultured in24-well FALCON plates at 37° C. in one mL of RPMI-1640 mediasupplemented with 2 g/L glucose, 0.3 g/L L-glutamate, and 2.0 g/LNaHCO₃, 15% heat-inactivated fetal bovine serum,1×penicillin/streptomycin, and 15 μL of hamster red blood cells whichhad been washed with RPMI-1640 medium. Five uL aliquots of the testcompounds in DMSO (prepared as above) or DMSO control were added to eachwell. The movement, feeding, and viability of the adult worms weremonitored for 24 hours. Afterwards, the media were removed and replacedwith fresh media to which the test compounds or DMSO had been added andthe adult worms observed for another 24 hours. Finally, the media wereagain removed and replaced with fresh media without the test compoundsor DMSO and the recovery of the adult worms was monitored for another 24hours.

At a concentration of 25 μg/mL, compounds 3 (2-hydroxychrysophanol) and6 (kwanzoquinone E) exhibited significant activity by completelyimmobilizing all cercariae within 15 seconds and 14 minutes,respectively. The dose effect of these compounds is shown in Table 5 andFIGS. 6 and 7.

TABLE 5 Percent of Cercariae Immobilized Concentration (μg/mL) Compound3 Error Compound 6 Error 0 0 0 0 0 1.56 40 10 40 10 3.125 90 4 40 106.25 96 2 40 10 12.5 100 0 92 2 25 100 0 94 2 Total of 10 assays foreach compound and 10 minutes per assay.The potency of compound 3 was not diminished even when diluted to aconcentration of 3.1 μg/mL. After 30 minutes of exposure to the testcompounds, the solution containing the test compounds was removed andreplaced with fresh media. Cercariae treated with compound 3 exhibited50% mortality after 24 hours while those exposed to compound 6 were alldead. None of the other compounds isolated from H. fulva roots,including glycosides of compounds 3 and 6, compounds 4 and 7,respectively, exhibited any activity at 25 μg/mL. The adult worms werealso immobilized within 16 hours by compounds 3 and 6 at 50 μg/mL.Following removal of the compounds, 35% and 55% of the adults exposed tocompounds 3 and 6, respectively, were dead. In contrast to the effectson the cercariae and adults, the intermediate schistosomula stage wasrefractory to all compounds at 35 μg/mL.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1. A method of inhibiting a parasitic helminth, which comprises:exposing the helminth to an antihelminthic amount of at least oneanthraquinone of the formula:

wherein R₁, R₂, R₃, and R₄ are each selected from the group consistingof hydrogen, hydroxy, halogen, alkyl, acetyl alkene, alkene, alkyne,aryl, cyclic, acid group, carbohydrate, and combinations thereof, R isselected from the group consisting of methyl, alkyl, aldehyde,hydroxymethyl, acid group, and carbohydrate, each R containing 1 to 12carbon atoms and the halogen is selected from the group consisting of I,F, Br, and Cl.
 2. The method of claim 2 wherein the anthraquinone hasthe formula:

wherein R is a group containing 1 to 12 carbons selected from the groupconsisting of methyl alkyl, acetyl, aldehyde, hydroxymethyl, acid, andcarbohydrate groups each containing 1 to 12 carbon atoms.
 3. The methodof claim 1 wherein the anthraquinone is 1,2,8-trihydroxy-3-methylanthraquinone.
 4. The method of claim 1 wherein the anthraquinone is1,2,8-trihydroxy-3-hydroxymethyl anthraquinone.
 5. The method of claim 1wherein the inhibition is in vitro.
 6. The method of claim 1 wherein theinhibition is in vivo.